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. 2021 May 27;11:11167. doi: 10.1038/s41598-021-90653-1

Figure 5.

Figure 5

Defects in the ICM lineage of embryos derived from single blastomeres. (a) Representative phase-contrast images showing the outgrowth of pseudoblastocysts derived from single blastomeres after 5 days in a blastocyst outgrowth assay. (b) Phenotypes of 1C, 2CB, 4CB, and 8CB pseudoblastocysts in the blastocyst outgrowth assay were classified into three categories, and their proportions are shown. The P values were determined with the two-sided Fisher’s exact test followed by adjustment with the BH method (FDR = 0.05). **P < 0.001. n indicates the number of pseudoblastocysts. All data were obtained from four replicate experiments. (c) 1C, 2CB, 4CB, and 8CB pseudoblastocysts that had been cultured in vitro up to 96 hpi were then cultured further for determination of their survival rate at 120 hpi (judged on the basis of a maintained cavity). The rate of survival is shown in the left panel, and the difference in survival rates among the different types of pseudoblastocyst and its simultaneous 95% CI are shown in the right panel. The P values were determined as in (b). **P < 0.001. n indicates the number of pseudoblastocysts. All data were obtained from three or four experiments performed on different days for each blastomere stage and were then analyzed. (d,e) 1C, 2CB, 4CB, and 8CB pseudoblastocysts cultured in vitro up to 120 hpi were subjected to bright-field imaging and to immunofluorescence staining with anti-NANOG and anti-GATA4 for evaluation of EPI and PrE formation. Representative images demonstrating the mutually exclusive expression of NANOG (red) and GATA4 (green) in two distinct compartments corresponding to EPI and PrE, respectively, of 1C, 2CB, and 4CB blastocysts are shown in (d). DNA was stained with Hoechst 33342 (blue). The phenotypes of pseudoblastocysts with regard to formation of EPI and PrE were classified into four categories, and their proportions are shown in (e). Embryos were obtained by in vitro fertilization on at least three different days. The P values were determined as in (b). **P < 0.001. n indicates the number of pseudoblastocysts.